Title
Structural and Functional Effects of Tryptophans Inserted into the Membrane-binding and Substrate-binding Sites of Human Group IIA Phospholipase A2
Author
Kathleen N. Nemec, Abhay H. Pande, Shan Qin, Ramona J. Bieber Urbauer, Shuhua Tan, David Moe, Suren A. Tatulian
Year
2006
Journal
Biochemistry
Abstract
Phospholipase A2 (PLA2) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val3 (membrane binding surface) and Phe5 (substrate binding pocket) of human group IIA PLA2 by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA2s upon membrane binding. Although PLA2 and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the α-helical structure presumably resulting from the tendency of Trp5 to insert into the membrane. Furthermore, whereas the PLA2 and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane−water interface. Although Trp3 at the membrane binding face of PLA2facilitates the proper membrane binding of the enzyme, Trp5 in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.
Full Article
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Ligand binding, Vesicle interactions, Fluorescence, Biochemistry